The long term goal of this work is to understand how substances diffuse in the brain extracellular space (ECS). The ECS is the narrow gap (about 30 - 50 nm) that separates one cell from another in brain. This gap contains a solution that resembles cerebrospinal fluid and an extracellular matrix made up of complex long chain carbohydrate molecules. This project focuses on the role of the extracellular matrix in hindering and patterning diffusion. The major tool for this work will be Monte Carlo simulation using the MCell software and this will be supported by experiments using the integrative optical imaging (IOI) method to measure extracellular diffusion in rat brain slices. The project has three Aims. The first Aim is to determine the origin of the extracellular components of anisotropic diffusion arising from aligned fiber bundles. Such bundles play a major role in the magnetic resonance technique of diffusion tensor imaging. Measurements of the extracellular diffusion of the probe ion tetramethylammonium have been made previously in three axes in both corpus callosum and in the molecular layer of the cerebellum. These measurements reveal paradoxes in the component values in both regions that suggest a more complex relation between geometry and diffusion than hitherto assumed. Modeling supported by experiments will determine the role of extracellular matrix and geometrical factors in accounting for the discrepancies. The second Aim will determine how the diffusion of growth factors is modified by binding to extracellular matrix. Growth factors are endogenous proteins that are potent agents that promote brain cell plasticity in adults and are essential during development. They diffuse in the ECS and are being trialed as therapeutic agents to relieve Parkinson's Diseases and other chronic illnesses. The extracellular matrix binds growth factors and may act as a local storage site for them and these processes are modified by the diffusion properties of the ECS geometry, however there is little quantitative understanding of these processes. Modeling and experiments with the FGF-2 growth factor will determine combinations of binding and diffusion and geometric parameters that will account for observed data. The third Aim is to improve the resolution of the IOI method which is based on imaging a cloud of diffusing molecules that are made visible by prior attachment of a fluorescent dye. Presently, the source of molecules is achieved by pressure injecting a small amount from a micropipette but in this project a highly controlled iontophoretic source will be developed to release the molecules. In some conditions, light scattering may limit the resolution of IOI and a correction for this will be formulated. PUBLIC HEALTH RELEVANCE: This project focuses on the role of the extracellular matrix (an entanglement of long complex molecules) in hindering diffusion of substances, including potential drugs, as they move in the narrow spaces between brain cells. Diffusion studies in fiber-rich brain regions will improve the basis for the magnetic resonance technique of diffusion tensor imaging (DTI) and investigations on the diffusion and binding of growth factors will facilitate therapeutic delivery to the brain. Development of an imaging technique for fluorescent molecules will provide a relatively inexpensive method for measuring diffusion of drugs in brain tissue.